This invention relates to relatively short peptides about 6-12 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogous to the naturally available peptides, and which include one or more D-tryptophan or D-leucine residues.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Mollusks of the genus Conus produce a highly toxic venom that enables them to carry out their unique predatory lifestyle. Prey are immobilized by the venom that is injected by means of a highly specialized venom apparatus, a disposable hollow tooth that functions both in the manner of a harpoon and a hypodermic needle.
Few interactions between organisms are more striking than those between a venomous animal and its envenomated victim. Venom may be used as a primary weapon to capture prey or as a defense mechanism. These venoms disrupt essential organ systems in the envenomated animal, and many of these venoms contain molecules directed to receptors and ion channels of neuromuscular systems.
The predatory cone snails (Conus) have developed a unique biological strategy. Their venom contains relatively small peptides that are targeted to various neuromuscular receptors and may be equivalent in their pharmacological diversity to the alkaloids of plants or secondary metabolites of microorganisms. Many of these peptides are among the smallest nucleic acid-encoded translation products having defined conformations, and as such, they are somewhat unusual. Peptides in this size range normally equilibrate among many conformations. Proteins having a fixed conformation are generally much larger.
The cone snails that produce these toxic peptides, which are generally referred to as conotoxins or conotoxin peptides, are a large genus of venomous gastropods comprising approximately 500 species. All cone snail species are predators that inject venom to capture prey, and the spectrum of animals that the genus as a whole can envenomate is broad. A wide variety of hunting strategies are used, however, every Conus species uses fundamentally the same basic pattern of envenomation.
Several peptides isolated from Conus venoms have been characterized. These include the .alpha.-, .mu.- and .omega.-which target nicotinic acetylcholine receptors, muscle sodium channels, and neuronal calcium channels, respectively (Olivera et al., 1985). Conopressins, which are vasopressin analogs, have also been identified (Cruz et al. 1987). In addition, peptides named conantokins have been isolated from the Conus geographus and Conus tutipa (Mena et al., 1990; Haack et al., 1990). These peptides have unusual age-dependent physiological effects: they induce a sleep-like state in mice younger than two weeks and hyperactive behavior in mice older than 3 weeks (Haack et al., 1990).
The standard amino acids in polypeptides translated from genes are exclusively in the L-configuration. In recent years it has been established that D-amino acids can be post-translationally introduced into such polypeptides (Kriel, 1994). Several small peptides have been characterized which contain a D-amino acid. The first of these was dermorphin, a potent heptapeptide agonist of the m-opiate receptor from amphibian skin, discovered by Erspamer and co-workers (Monteccuchi et al., 1981). A number of other peptides from amphibian skin (including the deltorphins and bombinin-H) were also found to have a D-amino acid. The cDNAs encoding these peptides were characterized (Richter et al., 1987; Richter et al., 1990). The results demonstrated unequivocally the presence of mRNA encoding the peptide precursor, indicating that the D-amino acid was post-translationally formed from the corresponding L-isomer.
In addition to these vertebrate systems, small peptides with D-amino acids have also been described in invertebrate systems, primarily molluscs. An FRMFamide analog from the bivalve, Mytilus edulis, which contains a D-leucine has been characterized (Fujisawa et al., 1992). Likewise, the land snail Achatina fulica has D-amino acid-containing small peptides, achatin-I and fulicin (Kamatani et al., 1989; Ohta et al., 1991). The cDNA encoding the precursor of fulicin was found to contain the usual L-Asn codon at the D-Asn position (Yasuda-Kamatani et al., 1995). Recently, the post-translational inversion of an amino acid was demonstrated in vitro for .omega.-agatoxin-IVB (also termed .omega.-agatoxin-TK), a Ca channel inhibitor from funnel web spider (Shikata et al., 1995). The peptide isomerase that preferentially acts on Ser.sup.46 of the 48-amino acid peptide has been isolated and characterized.
Although there is no homology between vertebrate and invertebrate peptides (and the three molluscan peptides exhibit no sequence similarity), in every case the D-amino acid is found in the second position. This suggests that for small D-amino acid-containing peptides, the proteolytic event which generates the mature peptide and the post-translational enzymatic system which converts an L- to a D-amino acid work in combination to always generate the D-amino acid at position 2.
Epilepsy is a recurrent paroxysmal disorder of cerebral function characterized by sudden brief attacks of altered consciousness, motor activity, sensory phenomena or inappropriate behavior caused by abnormal excessive discharge of cerebral neurons. Convulsive seizures, the most common form of attacks, begin with loss of consciousness and motor control, and tonic or clonic jerking of all extremities, but any recurrent seizure pattern may be termed epilepsy. The term primary or idiopathic epilepsy denotes those cases where no cause for the seizures can be identified. Secondary or symptomatic epilepsy designates the disorder when it is associated with such factors as trauma, neoplasm, infection, developmental abnormalities, cerebrovascular disease, or various metabolic conditions. Epileptic seizures are classified as partial seizures (focal, local seizures) or generalized seizures (convulsive or nonconvulsive). Classes of partial seizures include simple partial seizures, complex partial seizures and partial seizures secondarily generalized. Classes of generalized seizures include absence seizures, atypical absence seizures, myoclonic seizures, clonic seizures, tonic seizures, tonic-clonic seizures (grand mal) and atonic seizures. Therapeutics having anticonvulsant properties are used in the treatment of seizures. Most therapeutics used to abolish or attenuate seizures demonstrate activity at least through effects that reduce the spread of excitation from seizure foci and prevent detonation and disruption of function of normal aggregates of neurons. Anticonvulsants which have been utilized include phenytoin, phenobarbital, primidone, carbamazepine, ethosuximide, clonazepam and valproate. For further details of seizures and their therapy (see Rall & Schleifer (1985) and The Merck Manual (1992)).
It has been shown that neurotransmission mediated through the NMDA receptor complex is associated with seizures (Bowyer, 1982; MeNamara et al., 1988), ischemic neuronal injury (Simon et al., 1984; Park et al., 1988) and other phenomena including a synaptogenesis (Cline et al., 1987), spatial learning (Morris et al., 1986) and long-term potenitiation (Collinridge et al., 1983; Harris et al., 1984; Morris et al. , 1986). Regulation of these neuronal mechanisms by NMDA-mediated processes may involve activation of a receptor-gated calcium ion channel (Nowak et al., 1984; Mayer et al., 1987; Ascher and Nowak, 1988).
The NMDA channel is regulated by glycine. This amino acid increases NMDA-evoked currents in various tissues (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988) by increasing the opening frequency of the NMDA channel (Johnson and Ascher, 1987). Thus, NMDA-induced calcium influx and intracellular accumulation through may be stimulted by glycine (Reynolds et al., 1987; Wroblewski et al., 1989), which interacts with its own distinct site (Williams et al., 1991). Furthermore, accumulation of intracellular calcium may be implicated in the aforementioned neuropathologies.
It is desired to identify additional peptides which target the NMDA receptor. It is further desired to identify compounds which are useful as anticonvulsant or neuroprptective agents.